The present invention relates to the field of methods and compositions for directing human mesenchymal stem cells in vitro and in vivo to differentiate into chondrocytes prior to or at the time of, or after their implantation into a recipient or host for the therapeutic treatment of articular cartilage defects.
Mesenchymal stem cells (MSCs) are the formative pluripotent blast or embryonic-like cells found in bone marrow, blood, dermis, and periosteum that are capable of differentiating into specific types of mesenchymal or connective tissues including adipose, osseous, cartilaginous, elastic, muscular, and fibrous connective tissues. The specific differentiation pathway which these cells enter depends upon various influences such as mechanical influences and/or endogenous bioactive factors, including growth factors cytokines and/or local microenvironmental conditions established by host tissues.
A clonal rat fetus calvarial cell line has been shown to differentiate into muscle, fat, cartilage and bone (Goshima et al., Clin Orthop Rel Res. 269:274-283, 1991). Bone marrow cells form bone and cartilage following their encasement in diffusion chambers and in vivo transplantation (Ashton et al., Clin Orthop Rel Res. 151:294-307, 1980 (rabbit): Bruder et al., Bone Mineral. 11:141-151, 1990 (avian)). Cultured chick periosteum cells have been shown to differentiate into cartilage and bone in vitro (Nakahara et al., Exp. Cell Res., 195:492-503; 1991). Rat bone marrow-derived mesenchymal cells were shown to have the capacity to differentiate into osteoblasts and chondrocytes when implanted in vivo (Dennis et al., Cell Transpl, 1:2332, 1991; Goshima et al., Clin Orthop Rel Res. 269:274-283, 1991).
Chondrogenic differentiation of rabbit bone marrow derived mesenchymal progenitor cells has been studied in connection with articular cartilage healing utilizing cells in a pelleted format (Johnstone et al. Exp Cell Res 238(1):265-272 (1998).). However, cells in a condensed packed or pelleted cell mass do not have an optimal configuration in part due to the limitation on the maximum growth of the cells, limited permeability of nutrients, gases and growth factors, and other metabolic characteristics.
Pre-molded biodegradable multilayer matrices have been described for repair of articular cartilage, which have been packed into or press-fitted into regularly shaped osteochondral defects (Athanasiou U.S. Pat. No. 5,607,474). Cultured chondrocytes added to a collagen matrix for implantation into an articular cartilage lesion have also been described (Frenkel, S R et al., J Bone Joint Surg, 79-B(5):831-6 (1997)).
Alginate sponges have been used in studies of cartilage repair (see review Messner K. and J. Gilquist, Acta Orthop. Scand 67(5):523-529 (1996)). Mesenchymal cells from 12 day old mouse limb buds that were phenotypically undifferentiated but committed to differentiate to the chondrocytic lineage in an alginate bead culture system differentiated to cartilage cells and formed a pericellular matrix (Shakibaci, M. and P. De Souza, Cell Biology International. 21(2):75-86 (1997)). Adult human chondrocytes cultured in alginate beads formed a compartmentalized cartilage matrix (Hxc3xa4uselmann H J et al., Am. J. Physiol. 271 (Cell Physiol. 40):C742-C752, 1996). The growth of chondrocytes in alginate and collagen carrier gels has been compared (van Susante, J. et al., Acta Orthop Scand. 66(6):549-556 (1995)).
An optimized matrix to regenerate cartilage in vivo using mesenchymal stem cells is therefor required.
In accordance with one aspect of the invention, there is provided a construct which supports the differentiation and maturation of human mesenchymal stem cells into chondrocytes. In a preferred embodiment, the construct comprises human mesenchymal stem cells in association with a gel and preferably in an alginate suspension. The construct can be utilized for in vivo cartilage regeneration.
In a further aspect of the invention, soluble hyaluronic acid may be added to the construct to support chondrogenesis.
In accordance with another aspect, there is provided a composition for regenerating cartilage comprising human mesenchymal stem cells and an alginate gel. Preferably the matrix supports the differentiation and maturation of human mesenchymal stem cells into chondrocytes.
In one embodiment, the construct can be placed in vitro in culture media which will provide conditions favorable for chondrogenic differentiation of the MSCs in the gel. The constructs are cultured in this media and may be modified to determine the effect of specific agents on chondrogenic differentiation and/or the chondrocytic phenotype.
In another embodiment, the MSCs are added to the gel in vitro under conditions such that the MSCs attach to the gel to form an MSC-gel construct. The construct can then be placed in vivo, i.e. implanted at a target site. In this embodiment, the MSCs are not induced to differentiate into chondrocytes prior to implantation. When placed in vivo, the construct will be exposed to naturally occurring chondrogenic inducing factors, found for example in synovial fluid, to stimulate chondrogenic differentiation of the MSCs.
In a still further embodiment, the MSCs are added to the alginate solution and the MSC-alginate solution is placed in contact with chondrogenic medium in vitro for a period of time sufficient to direct the MSCs into the chondrogenic lineage. The culture period may be long enough to obtain either mature chondrocytes or may be interrupted at any stage of the chondrogenic differentiation. The entire construct or portions thereof may then be implanted into the defect site.
In another aspect of this embodiment, the MSCs are added to the alginate solution and the MSC alginate suspension is spread on a support. The alginate suspension is contacted with a CaCl2 solution. The alginate polymerizes and forms a gel layer encasing the MSCs. The layer may then be contacted with a chondrogenesis inducing factor.
For purposes of the present invention, the MSCs can be culture-expanded MSCs, freshly isolated MSCs or unpurified populations of MSCs. The MSCs may further be exposed to at least one chondroinductive agent.
Hyaluronic acid may be further added to the above embodiments to support chondrogenesis.
The invention also provides a process for producing chondrocytes from mesenchymal stem cells by contacting mesenchymal stem cells with a chondroinductive agent in vitro wherein the stem cells are associated with the alginate gel and then placed into the implant site.
The invention also provides a process for inducing chondrogenesis in mesenchymal stem cells by contacting mesenchymal stem cells with a chondroinductive agent in vitro wherein the stem cells are associated with an alginate gel. The culture period may be long enough to obtain either mature chondrocytes or may be interrupted at any stage of the chondrogenic differentiation. The entire construct or portion of the construct may be delivered to the defect site.
The invention further provides a method of repairing or regenerating damaged cartilage, comprising administering to an individual in need thereof a biocompatible construct comprising an alginate gel which supports the differentiation of human mesenchymal stem cells into the chondrogenic lineage.
The above methods can also preferably comprise steps where the cells are cultured with the chondroinductive composition and thereafter mixed in alginate gel suspension.
The above methods can further comprise steps where the cells are cultured with soluble hyaluronic acid and thereafter are mixed in alginate gel suspension.
In another embodiment of the present invention, the MSC-gel layer system may be delivered directly to the implant site without prior induction of differentiation of the MSCs to the chondrogenic lineage. In this embodiment the MSCs are allowed to attach to the gel for a period of up to 24 hours and then implanted without attempting to direct them into the chondrogenic lineage prior to implantation.
In an alternate embodiment, the MSC-loaded gel is placed into chondrogenic medium in vitro for a finite period to direct the MSCs into the chondrogenic lineage. The culture period may be long enough to obtain mature chondrocytes or may be interrupted at any stage of the chondrogenic differentiation. The entire construct or portions thereof may be delivered to the defect site.